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Cited by 37CRISPR and Genetic EngineeringRetinal Diseases and TreatmentsRetinal Development and Disorders

CRISPR-based VEGF suppression using paired guide RNAs for treatment of choroidal neovascularization

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Chung, Sook Hyun, Sin, Tzu-Ni, Dang, Brian, Ngo, Taylor, Lo, Therlinder, Lent-Schochet, Daniella, Meleppat, Ratheesh K., Zawadzki, Robert J. +1 more·2022·Molecular Therapy - Nucleic Acids
DOI 10.1016/j.omtn.2022.04.015PMC PMC9114159
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Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genomic disruption of vascular endothelial growth factor A ( Vegfa ) with a single gRNA suppresses choroidal neovascularization (CNV) in preclinical studies, offering the prospect of long-term anti-angiogenesis therapy for neovascular age-related macular degeneration (AMD). Genome editing using CRISPR-CRISPR-associated endonucleases (Cas9) with multiple guide RNAs (gRNAs) can enhance gene-ablation efficacy by augmenting insertion-deletion (indel) mutations with gene truncations but may also increase the risk of off-target effects. In this study, we compare the effectiveness of adeno-associated virus (AAV)-mediated CRISPR-Cas9 systems using single versus paired gRNAs to target two different loci in the Vegfa gene that are conserved in human, rhesus macaque, and mouse. Paired gRNAs increased Vegfa gene-ablation rates in human cells in vitro but did not enhance VEGF suppression in mouse eyes in vivo . Genome editing using paired gRNAs also showed a similar degree of CNV suppression compared with single-gRNA systems. Unbiased genome-wide analysis using genome-wide unbiased identification of double-stranded breaks (DSBs) enabled by sequencing (GUIDE-seq) revealed weak off-target activity arising from the second gRNA. These findings suggest that in vivo CRISPR-Cas9 genome editing using two gRNAs may increase gene ablation but also the potential risk of off-target mutations, while the functional benefit of targeting an additional locus in the Vegfa gene as treatment for neovascular retinal conditions is unclear.

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  • The efficacy of gene editing treatments for anti-VEGF is limited by delivery vehicles, poor efficiency of compact Cas9 nucleases, and risk of genomic alterations from long-term Cas9 expression.

    “However, the efficacy of such treatments is limited by the delivery vehicles, the poor efficiency of compact Cas9 nucleases, and the risk of genomic alterations from the long-term expression of Cas9 (46).”

    From Characterization of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina (2024) · cites this paper as [46]

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Cited by

Sorted by citations

Authors
Chung, S. H., Sin, T.-N., Dang, B., Ngo, T., Lo, T., Lent-Schochet, D., Meleppat, R. K., Zawadzki, R. J., & Yiu, G.
Affiliations
UC Davis Health (US)
Corresponding author
Yiu, Glenn
ORCID
Venue
Molecular Therapy - Nucleic Acids
Publisher
Elsevier BV
Published
2022-06
Volume
28
Pages
613-622
ISSN
2162-2531 (ISSN-L)
DOI
10.1016/j.omtn.2022.04.015
PMC
PMC9114159
OpenAlex
W4224952669
Cited by
37
References
10
Citations by year
2026: 9 · 2025: 7 · 2024: 12 · 2023: 8 · 2022: 1
FWCI
2.29
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Therapy — Nucleic Acids (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by-nc-nd)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Cancer Institute (P30CA093373)
  • National Eye Institute (R21 EY031108, R01 EY032238)
  • BrightFocus Foundation
  • Genentech
  • Macula Society
  • National Institutes of Health
  • University of California, Davis
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Health Sciences › Medicine › Ophthalmology › Retinal Diseases and Treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinal Development and Disorders
Keywords
CRISPR/Cas9, choroidal neovascularization, neovascular age-related macular degeneration, anti-angiogenic therapy, off-target effects, GUIDE-seq, indel mutations, rhesus monkeys, mouse eye

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